1. Field of the Invention
This invention relates to amplifying nucleic acids by thermal cycling and, more particularly, to automated machines for performing amplification reactions such as a polymerase chain reaction (PCR).
2. Description of the Related Art
DNA (Deoxyribonucleic acid) may be amplified by thermally cycling a specially constituted liquid reaction mixture according to protocol such as a polymerase chain reaction (PCR) protocol which includes several incubations at different temperatures. The reaction mixture is comprised of various components such as the DNA to be amplified (the target) and at least two oligonucleotide primers selected in a predetermined way so as to be complementary to a portion of the target DNA. The reaction mixture also includes various buffers, enzymes, and deoxyribonucleotide triphosphates, such as DATP, dCTP, dGTP, and dTTP. The duplex DNA molecule is denatured into two complementary single strands. The primers then anneal to the strands, and, in PCR, nucleoside monophosphate residues are then linked to the primers in the presence of an enzyme such as a thermostable DNA polymerase to create a primer extension product. After primer extension, twice as many duplex DNA molecules exist. This process is repeated, each time approximately doubling the amount of DNA present. The result is an exponential increase in the concentration of target DNA, known as "amplification" of the target DNA.
The polymerase chain reaction (PCR) has proven to be a phenomenal success for genetic analysis, largely because it is simple and very versatile, and requires relatively low cost instrumentation. A key to this success is the concept of thermal cycling: alternating steps of melting DNA, annealing short primers to the resulting single strands, and extending those primers to make new copies of the double stranded DNA.
The methodology of the polymerase chain reaction is more fully described in U.S. Pat. Nos. 4,683,202 and 4,683,195 which are hereby incorporated by reference.
The polymerase chain reaction (hereafter PCR) has been performed in disposable reaction tubes such as small, plastic microcentrifuge tubes or test tubes which are placed in an instrument containing a thermally controlled heat exchanger. Examples of these instruments are disclosed in U.S. Pat. No. 5,038,852, U.S. application Ser. No. 07/709,374, filed Jun. 3, 1991, and U.S. application Ser. No. 07/871,264, filed Apr. 20, 1992, all of which are hereby incorporated by reference in their entirety.
The heat exchanger in these instruments is typically a metal block; however, hot air ovens and water baths also have been used. The temperature of the reaction mixture in the reaction tubes is changed in a cyclical fashion to cause denaturation, annealing and extension reactions to occur in the mixture. Three separate incubation temperatures commonly were used in the first generation PCR thermal cycling applications. These were typically around 94.degree. C. for denaturation, around 55.degree. C. for annealing, and around 72.degree. C. for extension. More recently, the annealing and extension incubations have frequently been combined to yield a two temperature incubation process, typically around 94.degree. C. for denaturation, and around 50-65.degree. C. for an annealing and extension incubation. The optimal incubation temperatures and times differ, however, with different targets.
Rapid, small scale, PCR capillary tube instruments also have appeared. For example, Idaho Technology introduced an instrument wherein the reaction mixture is placed in capillary tubes which are then sealed and placed in a hot air oven which cycles the temperature of the reaction mixtures in the tubes. A similar system was described in a paper by Wittwer et al., "Minimizing the Time Required for DNA Amplification by Efficient Heat Transfer to Small Samples", Analytical Biochemistry 186, 328-331 (1990). There, 100 microliter samples placed in thin capillary tubes were placed in an oven with a heating coil, a solenoid activated door and a fan. Air was used as the heat transfer medium. A very similar system was also described by Wittwer et al in another paper entitled "Automated Polymerase Chain Reaction in Capillary Tubes with Hot Air," Nucleic Acids Research, Volume 17, Number 11, pp. 4353-57 (1989).
The PCR volume has been limited to a range of from about 10 microliters to 1.5 milliliters in conventional heat block or liquid bath heat exchanger PCR instrument designs where the reaction mixture has been stored in microcentrifuge tubes. It is hard to scale up these volumes. The difficulty resides in the fixed dimensions of the wells in the heat exchange block for the tubes and the escalating difficulty in achieving heat transfer uniformity among all wells as dimensions get larger and heat gradient problems become more pronounced. As the volume of prior art reaction vessels is increased, the surface/volume ratio decreases. This change reduces the ability to change quickly the temperature of the reaction mixture in each tube because most heat exchange occurs between the walls of the tubes and the walls of the wells in the sample block.
In prior art instruments, thermal ramps were long because there was substantial lag in the temperature of the sample relative to the block caused by poor convection and conduction. Substantial thermal ramp durations between incubation temperatures were often necessary to prevent significant temperature gradients from developing because of the large thermal mass of the metal blocks used in many instruments as well as nondiffuse heat sources and sinks. These temperature gradients can cause non-uniform amplifications in different samples located at diverse points along the temperature gradient. There is no chemical or biological reason for using temperature ramps.
A capillary tube PCR instrument has the advantage of rapid thermal incubation transitions because the reaction volume and sample containment thicknesses can be minimized. One such instrument is disclosed in U.S. Pat. No. 5,176,203, issued to D. M. Larzul. The Larzul patent discloses a wheel shaped apparatus for automatic thermal cycling of a fluid sample contained in a closed loop or spiral coil of continuous capillary tube. Each loop of the tube is routed through three thermostatted zones. The sample is pushed through the loops by a motorized magnetic system in which a magnet on the end of a rotating central arm magnetically pulls a slug of mineral oil containing suspended metallic particles through the capillary tube. Since the slug abuts the sample in the capillary tube, the slug pushes the sample through the loop. The motorized system may be micro-processor controlled to regulate the movement of the sample in accordance with a predetermined protocol.